Claudia González-Espinosa

Species heterogeneity of hepatic α1-adrenoceptors : α1A-, α1B- and α1C-subtypes

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.

Angiotensin II and active phorbol esters induce proto-oncogene expression in isolated rat hepatocytes

In isolated rat hepatocytes PMA, angiotensin II and to a lesser extent other hormones induce an early genetic response (increased expression of c-fos, c-mos, c-myc and beta-actin) without altering the expression of the glyceraldehyde 3-phosphate dehydrogenase gene. PMA, PDB and O-met-PMA, but not alpha-phorbol, stimulated c-fos expression. The effect of angiotensin II was inhibited by the AT1 antagonist, Losartan (DuP 753) (Ki approx. 25 nM), but not by the AT2 antagonist PD123177. Angiotensin II was much more effective than vasopressin or epinephrine in inducing proto-oncogene expression which suggests that angiotensin II receptors may exert actions in addition to those shared with the receptors for the other calcium-mobilizing hormones. The effect of PMA and angiotensin II on c-fos expression took place rapidly, with half times of 7 and 12 min, respectively. Actinomycin D markedly diminished basal c-fos expression whereas cycloheximide had the opposite effect. Actinomycin D diminished the effect of PMA and angiotensin II but it did not block them. PMA and the calcium-mobilizing hormones increased c-fos expression above the level observed with cycloheximide alone. These data suggest that PMA and the calcium-mobilizing hormones increased both transcription of the c-fos gene and stabilization of the proto-oncogene mRNA.

Characterization of the hepatic α1B-adrenoceptors of rats, mice and hamsters

The alpha 1-adrenoceptors present in liver membranes from rats, hamsters and mice were characterized using [3H]prazosin. In the liver membranes from the three species a relatively large number of receptors was observed (500-900 fmol/mg of protein) and the affinities for [3H]prazosin were very similar (0.2-0.3 nM). Membrane preincubation with 10 microM chloroethylclonidine markedly decreased [3H]prazosin binding and higher concentrations essentially abolished specific binding of this radioligand. Binding competition experiments indicated the following orders of potency: a) for agonists: oxymetazoline > epinephrine > or = norepinephrine >> methoxamine and b) for antagonists: prazosin > WB 4101 > or = phentolamine = benoxathian > 5-methyl urapidil. The affinity for (+)niguldipine was also low but there was variation between the three species. Total RNA obtained from the liver of these species hybridized with the alpha 1B-adrenergic cDNA probe. The data suggest that these receptors correspond to the alpha 1B subtype.

Hormonal modulation of c-fos expression in isolated hepatocytes. Effects of angiotensin II and phorbol myristate acetate on transcription and mRNA degradation

It has been shown that angiotensin II and PMA increase the expression of proto-oncogenes (c-fos, c-myc and c-mos) in liver cells. In this study the effects of angiotensin II and PMA on c-fos transcription and mRNA stability were investigated. Using nuclear run-off transcription assays, it was observed that PMA and angiotensin II induced a rapid increase in c-fos transcription. The transcription rate of the GAPDH gene did not change, indicating that the effects were not general on gene transcription. The ability of these agents to modulate proto-oncogene mRNA stability was tested by measuring c-fos mRNA half-life. It was observed that c-fos mRNA half-life was relatively short (approximately 14-18 min) and that angiotensin II and PMA markedly stabilized mRNA, increasing its half-life (approximately 4-fold and approximately 2-fold, respectively). The protein synthesis inhibitor cycloheximide increased mRNA stability to a much greater extent. Our results clearly demonstrate that angiotensin II and PMA increased c-fos mRNA accumulation in liver cells through two actions: induction of c-fos gene transcription and increase in mRNA stability.

Protein kinases and phosphatases modulate c-fos expression in rat hepatocytes. effects of angiotensin II and phorbol myristate acetate

In isolated rat hepatocytes angiotensin II and phorbol 12-myristate 13-acetate (PMA) induce the expression of c-fos. We studied the possible transduction pathway(s) involved in this effect using inhibitors of serine-threonine and tyrosine protein kinases. Calphostin and staurosporine, inhibitors of protein kinase C and other serine-threonine protein kinases, block in a dose-dependent manner the effect of angiotensin II and PMA. Interestingly, genistein also blocks the induction of this proto-oncogene, suggesting a role for tyrosine protein kinases. Inhibitors of serine-threonine protein phosphatases, such as okadaic acid, microcystin LR and calyculin also induce c-fos expression. These data suggest that protein phosphatases exert a tonic inhibitory control of c-fos expression. The effect of these phosphatase inhibitors were not blocked by staurosporine, calphostin or genistein. Our results suggest that the expression of c-fos in rat hepatocytes is regulated by complex phosphorylation-dephosphorylation cascade(s) probably involving serine/threonine and tyrosine protein kinase and protein phosphatase activities.

Angiotensin AT1 receptors in Clone 9 rat liver cells: Ca2+ signaling and c-fos expression

In C9 (Clone 9) liver cells, angiotensin 11 increased the intracellular Ca2+ content, inositol phosphate production and c-fos mRNA expression. Other angiotensins were also active with the order of potency being angiotensin II = angiotensin III >> angiotensin I > angiotensin IV. Losartan, but not PD 123177 (1-(4-amino-3-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imida zo [4,5c]pyridine-6-carboxylic acid), blocked the effects of angiotensin II. Pertussis toxin did not alter these actions of angiotensin II. These data indicate that the effects were mediated through angiotensin AT1 receptors involving pertussis toxin-insensitive G-proteins. Phorbol myristate acetate was also able to increase c-fos mRNA expression. The action of angiotensin II was consistently greater than that of the active phorbol ester. Staurosporine but not genistein inhibited this effect of angiotensin II. Angiotensin II- and phorbol myristate acetate-induced proto-oncogene mRNA expression was attenuated in cells incubated overnight with the active phorbol ester, which suggests a major role of protein kinase C.

Characterization of the α1-adrenoceptors of cat liver. Predominance of the α1A-adrenergic subtype

Abstract The α 1 -adrenoceptors present in the liver of cats were characterized using [ 3 H]prazosin. This radioligand binds to cat liver membranes with high affinity (K D , 0.79 nM) to a moderately abundant number of sites (160 fmol/mg of protein). This sites were characterized pharmacologically, by binding competition, observing the following orders of potency: a) for agonists: oxymetazoline > epinephrine = norepinephrine ⪢ methoxamine, and b) for antagonists: WB4101 ≥ prazosin ≥ (+) niguldipine ≥ benoxathian ≥ spiperone = 5-methyl-urapidil > phentolamine > BMY 7378. These data suggested that cat liver expresses α 1A -adrenoceptors. Expression of the mRNA for this receptor was confirmed by RT-PCR.

Inverse α1A and α1D adrenoceptor mRNA expression during isolation of hepatocytes

Abstract It is now well documented that changes in gene expression take place during cell isolation and culture. Here, we report the change in the expression of the mRNAs for α1-adrenoceptor subtypes, during dissociation of guinea pig liver cells with collagenase. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, it was observed that during the isolation procedure, the mRNA for the α1A-adrenoceptor, normally expressed in whole liver, was degraded and the mRNA for α1D subtype, barely expressed in whole liver, increased in an actinomycin D-sensitive manner. When the isolation procedure was performed in the presence of cycloheximide, the mRNA for the α1A-adrenoceptor did not diminish and the induction of the α1D-adrenoceptor mRNA was even more evident. Our data indicate that cell isolation alters α1-adrenoceptor mRNA expression.

Characterization of the α1-adrenoceptors of dog liver : predominance of the α1A-subtype

Abstract Using dog liver membranes we observed that [ 125 I]HEAT ((±)-β-([ 125 I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone) binds with high affinity ( K D 97 pM) to a discrete number of sites ( B max 40 fmol/mg protein) with the pharmacological characteristics expected for α 1 -adrenoceptors. Such sites were inactivated by pretreatment with chloroethylclonidine. Binding competition experiments indicated the following order of potency: (a) for agonists: oxymetazoline > epinephrine ≥ norepinephrine > methoxamine and (b) for antagonists: WB4101 ≥ 5-methyl-urapidil = prazosin ≥ benoxathian ≥ (+)-niguldipine > rmphentolamine. Northern analysis indicated that total RNA isolated from dog liver hybridized with an α 1c selective probe (bovine brain). The orders of potency for agonists and antagonists, their K i values and the Northern analysis suggest that dog liver expresses α 1A -adrenoceptors.

Coexpression of α1A- and α1B-adrenoceptors in the liver of the rhesus monkey (Macaca mulatta)

The alpha 1-adrenoceptors present in the liver of rhesus monkeys was characterized using [3H]prazosin. This radioligand binds to monkey liver membranes with high affinity (KD 0.33 nM) to a moderately abundant number of sites (97 fmol/mg of protein). These sites were characterized pharmacologically, by binding competition, observing two affinities for most ligands. The order of potency for agonists was: (a) for the high affinity sites: oximetazoline > epinephrine = norepinephrine > methoxamine; and (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective agonists): oximetazoline > or = epinephrine = norepinephrine > > methoxamine. For antagonists the orders of potency were: (a) for the high affinity sites: R-(-)-5[2-[[2-(ethoxyphenoxy)ethyl]amino]propyl]-2-metoxybenzen esulfonamide HCl (tamsulosin) > or = 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane (WB4101) > or = prazosin > or = (+)-niguldipine > 5-methylurapidil = benoxathian > phentolamine > 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]deca ne-7,9- dione dihydrochloride (BMY 7378); (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective antagonists): prazosin > tamsulosin > phentolamine = WB4101 > (+)-niguldipine > or = 5-methyl-urapidil = benoxathian > BMY 7378. These data strongly suggest that Macaca mulatta liver cells coexpress alpha 1A- and alpha 1B-adrenoceptors. Expression of the mRNA for these receptors was confirmed by reverse transcriptase-polymerase chain reactions.